Recently, it has been found that a brain and a spinal cord have neural stem/progenitor cells, and it has been reported that ES (embryonic stem) cells are differentiated into specific central nervous system cells. This has raised expectations for central nervous system regenerative medical treatment. Further, as a process for isolating and selectively culturing the neural stem/progenitor cells, a neurosphere method (floated coagulated mass culturing technique) has been established. Furthermore, a method for inducing differentiation of the neural stem/progenitor cells has been reported which is designed to induce differentiation of the neural stem/progenitor cells into neural cells by culturing a sphere (aggregate) made from the neural stem/progenitor cells by adherent culturing after the floated coagulated mass culturing technique.
Further, it has been reported that by transplanting into a living organism neural stem/progenitor cells derived from a brain or a spinal cord, or ES cells, the transplanted cells are differentiated into specific nerve cells as a result of adaptation to the environment (see Non-Patent Document 1: Nature 414, p. 112-117, review, 2001).
Incidentally, when retinal nerve cells of a mammal are once degenerated, the retinal nerve cells cannot be regenerated, so that functions of the retinal nerve cells are impaired. Furthermore, degeneration of visual cells due to a retinal degeneration disease or the like may lead to loss of sight. At present, there is no effective treatment for such an intractable disease. Accordingly, if the retinal nerve cells can be produced by differentiation induction of the neural stem/progenitor cells as described above, a highly effective regenerative medical treatment will be realized.
As the neural stem/progenitor cells used to induce differentiation into the retinal nerve cells, ciliary epithelial cells and retinal pigmented epithelial cells have been used so far. The retinal pigmented epithelial cells and the ciliary epithelial cells are derived from neural plates. The photoreceptor layer is coated with the retinal pigmented epithelial cells. The photoreceptor layer is an outermost cell layer of a retina including a plurality of cell layers. A ciliary epithelium is tissue which lies between an iris and a retina.
For example, reported in Non-Patent Document 2 (Science 287, p. 2032-2035, 2000) is a method for inducing differentiation by culturing, according to the adherent culturing, spheres (aggregates) which have been formed by culturing ciliary epithelial cells according to the floated coagulated mass culturing technique. Also in Non-Patent Document 3 (Biochem. and Biophys. Res Commun. 270, p517-521, 2000), it has been reported that there is a possibility that differentiation of the ciliary epithelial cells into retinal nerve cells can be induced.
Further, in Non-Patent Document 4 (Brain Res. 677 (2), p. 300-310, 1995), it is reported by a culturing experiment that retinal pigmented epithelial cells of a mammal are differentiable into nerve cells, albeit in a limited portion of a fetal period. Note that, also in Non-Patent Document 5 (DNA Cell Biol 12 (8), p. 667-673, 1993), it has been reported that there is a possibility that differentiation of retinal pigmented epithelial cells into retinal nerve cells can be induced.
If neural stem/progenitor cells, derived from a brain or a spinal cord, and ES cells, are used to regenerative medical treatment, the use raises many problems such as immunological rejection caused by cell transportation, ethical issues, and unbalance between demand and supply of transplant cell sources. If it becomes possible to use, as a transplant source, cells derived from a transplant recipient per se, it will pave a way to autogenous transplantation, thus solving the foregoing problems.
However, in view of medical applications, an idea of using ciliary epithelial cells as materials for central nervous system regeneration is unrealistic, because it is very difficult to obtain the ciliary epithelial cells from a patient per se.
Further, Non-Patent Document 4 states that, for mammals, differentiability into nerve cells is observed only in retinal pigmented epithelial cells in a limited time within a fetal period in which many relatively undifferentiated cells exist. That is, relatively-undifferentiated cells rarely exist in tissue of an adult mammal. Since retinal pigmented epithelial cells of an adult is also highly differentiated, it is difficult to isolate and culture the retinal pigmented epithelial cells. Therefore, at present, it is impossible to use the retinal pigmented epithelial cells of the adult mammal as materials for central nervous system regeneration.
Further, although a method for producing retinal nerve cells from ES cells is reported in Non-Patent Document 6 (Biochem. and Biophys. Res. Commun. 297, p. 117-184, 2000), the method is extremely inefficient.
Under such circumstances, iris pigmented epithelial cells of an eyeball are one example of cells expected to serve as materials for central nervous system regeneration. The iris pigmented epithelial cells are a component of an iris that opens and narrows a pupil in accordance with an amount of light so as to adjust an amount of light which reaches a retina. Like the retinal pigmented epithelial cells and the ciliary epithelial cells, the iris pigmented epithelial cells are derived from neural plates. Since it is sufficiently possible to collect part of iris pigmented epithelial cells from a patient per se, the iris pigmented epithelial cells can be effectively utilized as regenerative materials capable of autologous transplantation.
It has been deemed difficult to isolate and culture iris pigmented epithelial cells due to a small number of iris pigmented epithelial cells and a small number of tissues formed therefrom. However, the inventors have reported that the inventors have successfully isolated and cultured iris pigmented epithelial cells of a chick (Non-Patent Document 7: Experimental Cell Res. 245, p. 245-251, 1998). In Non-Patent Document 7, a culturing experiment shows that iris pigmented epithelial cells of the chick are capable of being differentiated into lenses.
Furthermore, the inventors have made it possible to isolate and culture iris cells of a mammal (mouse, rat, or human embryo) by a method improved from the process of Non-Patent Document 7 (see Non-Patent Document 8: Nature Neuroscience 4 (12), p. 1163, 2001).
In Non-Patent Document 8, primary culturing of isolated iris tissue of an adult rat was carried out, and although it was confirmed that some cells expressed neural markers, no specifically differentiated neural marker was detected. It was confirmed that the cultured iris cells form a rhodopsin protein necessary for a photoreceptor function, when in order to obtain visual cells of a retina, the cultured iris cells were forced to express Crx gene, which is suggested to play an important role in a period during which the visual cells are generated.
In Non-Patent Document 8, differentiation into the visual cells is induced only in the case where Crx gene, which is a specific gene, is expressed. However, when medical applications are taken into consideration, it is not preferable to induce differentiation by expressing a gene because inducing differentiation by expressing a gene involves a risk of damaging DNA.
Therefore, at present, no method has been established which, by inducing differentiation of neural stem/progenitor cells (iris pigmented epithelial cells) derived from iris tissue, produces retinal nerve cells which can be effectively used for regenerative medical treatment.
As described above, it is possible to collect part of iris pigmented epithelial cells from a patient per se. Therefore, if retinal nerve cells are obtained by inducing differentiation of iris pigmented epithelial cells, then regenerative medical treatment which uses cells of a patient per se will be realized. Moreover, it is expected that an important contribution will be brought about to establishing treatment for a retinal degeneration disease for which there is no effective treatment at present.
The present invention has been completed in consideration of the foregoing problems and has as an object to provide a method for producing retinal nerve cells by inducing differentiation into retinal nerve cells, without requiring gene transfer, from iris pigmented epithelial cells which may be effectively used for regenerative medical treatment, and the retinal nerve cells obtained by the method.